Hopes Raised by HDAC Inhibitor, but Uncertainties Remain

This past Wednesday in the journal Nature, Nancy Archin and colleagues from the laboratory of David Margolis published results from a trial investigating whether the approved cancer drug vorinostat can reverse HIV latency. Vorinostat (also known as SAHA, trade name Zolinza) belongs to a group of compounds called histone deacetlyase (HDAC) inhibitors. Histone deacetylases are a class of cellular enzymes involved in condensing DNA and repressing gene expression. In laboratory experiments, inhibiting histone deacetylases appears to free latent HIV from lockdown, causing viral RNA to be produced. HDAC inhibitors have therefore emerged as lead candidates for depleting latent HIV reservoirs, a task that is widely believed to be an important step along the path to a cure.

Margolis has previously presented preliminary results from the trial, first at a meeting in St. Maarten in December 2011, then at CROI and Keystone in March of this year. Each time the number of participants with available data increased, from four initially to six at CROI and seven at Keystone. The published Nature paper brings the final count to eight. I wrote about the data when it was initially presented, and below is an updated version of that report.

The first step of the protocol involved screening potential participants to assess whether vorinostat could reactivate latent HIV from their CD4 T cells ex vivo. Sixteen individuals had lymphocytes extracted by leukopheresis, then sorted into discrete pools of 1 million purified resting CD4 cells each (ending up with 12-48 pools per participant). These pools were exposed to either vorinostat or no drug, and a mean level of HIV RNA per million cells (and a standard deviation) was calculated for each person (the assay used can measure down to 10 copies per million cells). Margolis noted in St. Maarten that the statistical approach used to calculate the mean RNA levels is robust but complicated, and a paper explaining it is currently in press at a statistics journal. Eleven of the sixteen people screened showed an upregulation of HIV RNA expression in this analysis and eight agreed to participate in the next step of the trial.

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A 200mg dose of vorinostat was given first for safety, followed by a 400mg dose to study pharmacokinetics and for analyses of histone acetylation and acetylation of the p21 gene (in other words, analyses of the effects of the drug on cellular genetic machinery and not HIV). The pharmacokinetic data mirrored reports from cancer studies, with peak levels occurring a mean two hours after dosing. A significant increase in the acetylation of cellular histone H3 was observed, along with a trend towards increased acetylation of histones at the p21 gene in five participants who had sufficient cells available for this analysis.

A final 400mg dose of vorinostat was then administered with leukopheresis performed 4-6 hours afterward based on the pharmacokinetic data indicating this would be around the time of maximum activity. No grade 1 or greater toxicities were seen, and cell-associated unspliced HIV gag RNA expression increased significantly compared to baseline in all eight individuals by a mean of 4.8-fold (range: 1.5-10-fold). HIV RNA in peripheral blood was assessed using a single copy assay but no change was detected. The researchers also state that "a limited evaluation did not reveal a substantial reduction in the frequency of replication-competent HIV within resting CD4 T cells" but the data is not included in the paper, and they note that this would not be unexpected given that only a single dose was administered. The primary import of the results, they conclude, is the proof-of-concept: "these findings demonstrate that therapy targeted at persistent, latent infection within resting CD4 T cells is feasible, and open the way for the development of HDAC inhibitors with improved specificity, potency and safety profiles for the selective targeting of latent proviral genomes."

When he presented the early results in December 2011, Margolis offered a list of questions that remain to be answered.

Is there an equal effect from multiple doses or does it become attenuated?

How much drug exposure is needed?

Should drug be administered continuously or pulsed?

Will toxicities emerge?

What number of cells is needed to measure relatively rare reactivation events?

Are additional inducers needed?

Does RNA expression lead to virion production or clearance of the infected cell?

Are additional interventions needed to clear the latently cells that have been induced to express HIV RNA?

In addition to the caveat that HDAC inhibitors alone appear unlikely to be sufficient to achieve a cure of HIV infection, there is also a question that wasn't on Margolis's list: are there latently infected cells that cannot be reactivated by these drugs? At a symposium on cure research that took place immediately prior to AIDS 2012, Robert Siliciano presented evidence that, while the majority of resting CD4 T cells containing HIV DNA harbor viral genomes that are compromised by various genetic alterations (such as deletions) some -- 16.8% in his study (range: 6-36%) -- contain replication-competent HIV that is not induced by normal methods. It is as yet unclear if this virus can be induced to replicate in vivo.

In a commentary accompanying the Archin paper, Steve Deeks highlights the work that remains and the likely need for combination approaches, but also emphasizes that these results represent an important first step beyond antiretroviral therapy and into new territory, with a cure for HIV infection the ultimate destination.

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